125I-vasoactive Intestinal Peptide Research Articles

Brain delivery of vasoactive intestinal peptide enhanced with the nanoparticles conjugated with wheat germ agglutinin following intranasal administration

The development of biotech drugs such as peptides and proteins that act in the central nervous system has been significantly impeded by the difficulty of delivering them across the blood-brain barrier. The surface engineering of nanoparticles with lectins opened a novel pathway to the absorption of drugs loaded by biodegradable poly (ethylene glycol)-poly (lactic acid) nanoparticles in the brain following intranasal administration. In the present study, vasoactive intestinal peptide, a neuroprotective peptide, was efficiently incorporated into the poly (ethylene glycol)-poly (lactic acid) nanoparticles modified with wheat germ agglutinin and the biodistribution, brain uptake and neuroprotective effect of the formulation were assessed. The area under the concentration-time curve of intact 125I-vasoactive intestinal peptide in brain of mice following the intranasal administration of 125I-vasoactive intestinal peptide carried by nanoparticles and wheat germ agglutinin-conjugated ones was significantly enlarged by 3.5 approximately 4.7 folds and 5.6 approximately 7.7 folds, respectively, compared with that after intranasal application of 125I-vasoactive intestinal peptide solution. The same improvements in spatial memory in ethylcholine aziridium-treated rats were observed following intranasal administration of 25 microg/kg and 12.5 microg/kg of vasoactive intestinal peptide loaded by unmodified nanoparticles and wheat germ agglutinin-modified nanoparticles, respectively. Distribution profiles of wheat germ agglutinin-conjugated nanoparticles in the nasal cavity presented their higher affinity to the olfactory mucosa than to the respiratory one. Inhibition experiment with specific sugars suggested that the interaction between the nasal mucosa and the wheat germ agglutinin-functionalized nanoparticles were due to the immobilization of carbohydrate-binding pockets on the surface of the nanoparticles. The results clearly indicated wheat germ agglutinin-modified nanoparticles might serve as promising carriers especially for biotech drugs such as peptides and proteins.

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes.

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.

Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells.

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.

Retinoic acid regulation of the VIP and PACAP autocrine ligand and receptor system in human neuroblastoma cell lines.

Neuroendocrine tumors, neuroblastoma in particular, commonly express the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) and their receptors. Retinoic acid (RA) has been shown to induce differentiation of neuroblastoma cell lines, possibly by augmenting or interfering with neuropeptide autocrine loops. We sought to determine which receptor gene subtypes are expressed in selected human neuroblastoma cell lines (SH-SY5Y, IMR-32, and LA-N-5), and the effect of RA on the VIP/PACAP ligand/receptor system. Expression of both PACAP1 and VIP1/PACAP2 receptor genes was detected by Northern analysis, which characteristically encode Type I (PACAP-preferring), and Type II (bivalent VIP/PACAP) receptors, respectively. Binding experiments carried out on IMR-32 cells, using 125I VIP and 125I PACAP-27 as tracers, corroborated that both receptor subtypes were expressed. In contrast to RA upregulation of VIP binding (confirmed here in IMR-32 cells), levels of both receptor mRNAs were reduced after RA treatment. VIP mRNA in each cell line was increased by RA, whereas PACAP mRNA, detected in IMR-32 cells only, was reduced. The studies indicate that several components of the VIP/PACAP autocrine system are regulated in neuroblastoma cell lines during RA differentiation.

Glaucoma, capillaries and pericytes. 3. Peptide hormone binding and influence on pericytes.

To test the potential for vasoactive neuropeptide receptors to affect capillary resistance, we have begun to study the plausibility that pericytes might be equipped to respond to a representative peptide vasoconstrictor and a representative peptide vasodilator. Pericytes cultured from the bovine retinal vasculature specifically bind the angiotensin II (Ang II) antagonist saralasin (1 nM125I-saralasin bound at 2.2 +/- 0.41 fmol/mg protein) and 125I-vasoactive intestinal peptide (VIP; Kd of 0.5 nM with a population of 30 fmol/mg protein). Incubation with 100 microM Ang II induced minimal cAMP synthesis, while VIP (1 microM, 10 microM) did not induce any change in cAMP concentration. Ang II (10 microM and 100 microM) caused contraction of pericytes cultured on an elastic silicone surface. Circulating or locally produced vasoactive neuropeptides might affect pericyte contractile tone via several intracellular pathways, moderated by indirect effects of these peptides through endothelial stimulation, with the net effect on local blood flow resulting from the effects on arteries and veins as well as capillaries.

Chronic ethanol intake inhibits both the vasoactive intestinal peptide binding and the associated cyclic AMP production in rat enterocytes

1. 1. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of 125I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. 2. Native peptide displaced 125I-VIP binding to enterocytes, exhibiting a IC 50 at about 4 nM native VIP in control and ethanol-treated animals. 3. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-affinity and the low-affinity binding sites. 4. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.

Carbachol-induced down-regulation of high-affinity receptors for vasoactive intestinal peptide

When dispersed acini from guinea pig pancreas are first incubated with carbachol, the subsequent binding of 125I-vasoactive intestinal peptide (VIP) is inhibited during a second incubation. This inhibitory action of carbachol on binding of 125I-VIP depends on time, temperature, and the concentration of carbachol in the first incubation and can be blocked by atropine. First incubating acini with A23187, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), cholecystokinin octapeptide, bombesin, or 12-O-tetradecanoylphorbol-13-acetate does not alter binding of 125I-VIP. Adding EGTA to the first incubation medium abolishes the effect of carbachol on binding of 125I-VIP. In control acini or acini first incubated with carbachol, approximately half of the bound 125I-VIP can be stripped by acetic acid. 125I-VIP interacts with two distinct classes of receptors on pancreatic acini. One has a high affinity for VIP (Kd, 1 nM); the other has a low affinity for VIP (Kd, 2 microM). First incubating acini with carbachol decreases the number but not the affinity of high-affinity VIP receptors with no change in the number or affinity of low-affinity VIP receptors. Pancreatic acini possess two classes of muscarinic cholinergic receptors: one has a high affinity (Kd, 4 microM) and the other has a low affinity (Kd, 698 microM) for carbachol. The dose-response curve for carbachol-induced inhibition of binding of 125I-VIP and that for occupation of low-affinity muscarinic cholinergic receptors by carbachol are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of vasoactive intestinal peptide and secretin on chief cells prepared from guinea pig stomach.

Vasoactive intestinal peptide and secretin increased cellular cAMP and pepsinogen secretion in dispersed chief cells from guinea pig gastric mucosa. With each peptide there was a close correlation between the dose-response curve for changes in cellular cAMP and that for changes in pepsinogen secretion. Vasoactive intestinal peptide-(10-28) and secretin-(5-27) had no agonist activity and antagonized the actions of vasoactive intestinal peptide and secretin on cellular cAMP and pepsinogen secretion. Studies of binding of 125I-vasoactive intestinal peptide and of 125I-secretin indicated that gastric chief cells possess four classes of binding sites for vasoactive intestinal peptide and secretin and that occupation of two of these classes of binding sites correlates with the abilities of vasoactive intestinal peptide and secretin to increase cellular cAMP and pepsinogen secretion. What function, if any, is mediated by occupation by the other two classes of binding sites remains to be determined.

Preferential binding of vasoactive intestinal peptide to hepatic nonparenchymal cells.

The binding of 125I-vasoactive intestinal peptide (125I-VIP) and 125I-insulin has been examined in highly enriched populations of rat hepatocytes and hepatic nonparenchymal cells. 125I-VIP bound to high-affinity sites (Ka = 1.7 X 10(9) M-1) in nonparenchymal cells. Specific binding in these cells was nearly fivefold greater than in hepatocytes (15.0 +/- 0.6% vs. 3.6 +/- 0.7% radioactivity bound per 4 X 10(5) cells). In contrast, 125I-insulin binding was similar in both cell populations (18.2 +/- 2.4% per 4 X 10(5) cells in hepatocytes vs. 17.1 +/- 1.0% in nonparenchymal cells). Glucagon and insulin had no effect on 125I-VIP binding in nonparenchymal cells. Secretin inhibited 125I-VIP binding but was only about 1% as potent as unlabeled VIP. VIP had no apparent effect on cAMP levels in either cell population, whereas glucagon increased cAMP levels in both cell types. Our findings suggest that VIP binds preferentially to hepatic nonparenchymal cells and that these cells are primarily responsible for the clearance of VIP from the portal circulation.

Receptors for vasoactive intestinal peptide and secretin on rat pancreatic acini.

In dispersed acini from rat pancreas, binding of 125I-labeled vasoactive intestinal peptide and 125I-labeled secretin was relatively rapid, reversible, saturable, and temperature dependent. The rate of dissociation of bound 125I-labeled peptide was not a function of the concentration of free vasoactive intestinal peptide or secretin, indicating that the apparent affinities of these labeled peptides for their binding sites do not depend on the extent of receptor occupation. Four classes of receptors are required to account for the actions of vasoactive intestinal peptide and secretin on enzyme secretion, cellular cAMP, and binding of 125I-vasoactive intestinal peptide and 125I-secretin. One class has a high affinity for vasoactive intestinal peptide, and occupation of this class of receptors causes increased cellular cAMP and stimulation of amylase secretion. A second class has a low affinity for vasoactive intestinal peptide and for secretin, and occupation of these receptors does not cause changes in cAMP or amylase secretion. A third class of receptors has a high affinity for secretin, and occupation of these receptors causes increased cAMP and stimulation of amylase secretion. A fourth class of receptors has a low affinity for secretin, and occupation of these receptors causes stimulation of amylase secretion by a non-cAMP-mediated mechanism.

Action of natural glucagon on pancreatic acini: due to contamination by previously undescribed secretagogues.

In dispersed acini from guinea pig pancreas, natural glucagon caused a two- to threefold increase in amylase secretion, and this natural glucagon-induced increase was augmented by theophylline. Natural glucagon also caused a sixfold increase in cellular cAMP but did not alter cellular cGMP or outflux of 45Ca. Natural glucagon caused detectable changes in cAMP and amylase secretion at a concentration of 1 microM, half-maximal stimulation at 10 microM, and maximal stimulation at 100 microM. Natural glucagon potentiated the increase in enzyme secretion caused by secretagogues that act by causing mobilization of cellular calcium but did not alter the increase in enzyme secretion caused by secretagogues that increase or mimic the action of cellular cAMP. At concentrations up to 100 microM, natural glucagon did not inhibit binding of 125I-vasoactive intestinal peptide. The potency with which glucagon stimulated amylase release and augmented the increase in amylase release caused by cholecystokinin or carbachol was the same with acini from rat or mouse pancreas as it was with acini from guinea pig pancreas. Biologically active synthetic glucagon did not increase enzyme secretion. On reverse-phase, high-pressure liquid chromatography of natural glucagon, the ability to stimulate enzyme secretion migrated differently from the glucagon. Natural glucagon, at concentrations that stimulated cAMP accumulation, did not react with vasoactive intestinal peptide or secretin radioimmunoassays. Neither insulin nor pancreatic polypeptide, which are known to contaminate natural glucagon preparations, increased enzyme secretion from pancreatic acini. Trypsin treatment abolished the ability of natural glucagon to increase enzyme secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of Gila monster venom on dispersed acini from guinea pig pancreas.

Venom from Gila monster (family Helodermatidae) contains a pancreatic secretagogue. In dispersed acini from guinea pig pancreas, the venom increased enzyme secretion to the same extent as did vasoactive intestinal peptide, secretin, or PHI. The abilities of vasoactive intestinal peptide and Gila monster venom to stimulate enzyme secretion were not altered by boiling but were abolished by incubation with trypsin or chymotrypsin. Like vasoactive intestinal peptide, secretin, and PHI, the venom caused a 50- to 60-fold increase in cellular cAMP and inhibited binding of 125I-vasoactive intestinal peptide to its membrane receptors on pancreatic acini. The action of venom on enzyme secretion was inhibited by [Gln9]secretin-(5-27), a vasoactive intestinal peptide receptor antagonist, but was not altered by atropine, a cholinergic receptor antagonist, or by dibutyryl cGMP, a cholecystokinin receptor antagonist. Gila monster venom contained no immunoreactive vasoactive intestinal peptide by radioimmunoassay. These results indicate that venom from Gila monster contains a peptide that can stimulate pancreatic enzyme secretion by interacting with vasoactive intestinal peptide receptors on pancreatic acinar cells and thereby activating adenylate cyclase and increasing cellular cAMP.

Actions of a newly isolated intestinal peptide PHI on pancreatic acini.

In dispersed acini from guinea pig pancreas, PHI, a peptide recently isolated from porcine intestine and found to contain 27 amino acids, inhibited binding of 125I-vasoactive intestinal peptide (125I-VIP), increased cellular cAMP, and stimulated amylase secretion. The increase in amylase secretion caused by a maximally effective concentration of PHI in combination with 8-bromo-cAMP, VIP, or secretin was the same as that caused by PHI alone. In contrast, the increase in amylase secretion caused by PHI plus bombesin, carbachol, or the C-terminal octapeptide of cholecystokinin was significantly greater than the sum of the increase caused by each secretagogue acting alone. From the abilities of PHI to inhibit binding of 125I-VIP, to increase cellular cAMP, and to increase amylase secretion, the apparent affinity of PHI for the VIP-preferring receptors on pancreatic acinar cells is approximately 25 times less than that of VIP but 10 times greater than that of secretin. From the ability of PHI to increase cellular cAMP, the apparent affinity of PHI for the secretin-preferring receptors on pancreatic acinar cells is approximately 300 times less than that of secretin but equal to that of VIP.

Interaction of vasoactive intestinal peptide with a cell line (HeLa) derived from human carcinoma of the cervix: binding to specific sites and stimulation of adenylate cyclase.

The binding of vasoactive intestinal peptide (VIP) and its effect on cyclic AMP production were assessed in HeLa cells. The binding of [125I]VIP is a moderately rapid process, reversible, saturable, specific and dependent on temperature. Virtually no inactivation of the peptide is observed after 2 h of exposure to the cells. At 15 degrees C, the binding data obtained at steady state are compatible with the existence of two classes of binding sites: a first class with a Kd of 2.4 nM and low binding capacity (1.5 X 10(5) sites/cell) and a second class with a Kd of 100 nM and a high binding capacity (4.9 X 10(6) sites/cell). Secretin is eight times less potent than VIP in competing with 125I VIP but glucagon, insulin and somatostatin are inactive. VIP-induced stimulation of cyclic AMP production depends on time and temperature and is potentiated by a phosphodiesterase inhibitor. A concentration of VIP as low as 10(-10) M is able to stimulate adenylate cyclase. Half-maximal stimulation is observed at 10(-9) M and maximal stimulation (4 times above basal levels) at 10(-8) M VIP. Secretin is an agonist of VIP but exhibits a 1000 times lower potency with respect to adenylate cyclase activation. Glucagon, insulin and somatostatin do not show any effect. The presence of high-affinity binding sites and high sensitivity and specificity of adenylate cyclase for VIP in HeLa cells provide a good model to study the role of this peptide on cell proliferation and differentiation.